清毒栓干预宫颈HR-HPV感染局部免疫微环境的实验及临床研究

【作者】 楼姣英；

【导师】 金哲；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 目的1.通过检测宫颈上皮内瘤变组织局部免疫微环境相关转录因子T-bet、GATA3、Foxp3的免疫组化表达,从细胞水平探讨宫颈上皮内瘤变病程进展与病变局部免疫微环境中Th1、Th2、Treg细胞数量变化的关系2.以SiHa宫颈癌细胞上清液、CTL、Treg淋巴细胞共培养体系作为研究对象,观察Treg、清毒栓对CTLA-4、PD-1的蛋白表达的影响,探索Treg细胞在肿瘤免疫逃逸中的机制,探讨中药清毒栓对局部免疫微环境的作用。3.通过检测清毒栓、安达芬及随诊观察三组经治疗观察3个月后病毒负荷及临床症状积分的变化,判断中药清毒栓的临床疗效。方法实验研究：1.选择经HE染色,病理明确诊断为宫颈炎症及宫颈上皮内瘤变的宫颈组织128例,分为宫颈炎症、CINⅠ级、CINⅡ级及CINⅢ级4组,其中宫颈炎症、CINⅡ级病变组织32例,CINⅠ级34例及CINⅢ级病变组织30例。用免疫组化法检测各组组织中免疫微环境相关转录因子T-bet、GATA3、Foxp3阳性细胞的浸润数量,分析以上三种免疫细胞数量变化与病变进展的关系。2.CCK-8法测定PBMC生长曲线。3.用不同浓度SiHa细胞培养上清液作用于CTL细胞及Treg细胞,流式细胞术检测CD8、Treg细胞比例变化。4.免疫磁珠法分离人外周血CD8+T细胞、Treg细胞,流式细胞术检测分选后细胞的纯度。5.用CFSE标记法检测淋巴细胞增值,了解Treg细胞对CTL细胞的抑制功能。6.CCK8法测定各浓度β-榄香烯、清毒栓对CTL细胞增殖的影响。7.应用Western-Blot检测并比较CTL正常培养组(对照组)、CTL+50%SiHa上清培养组(癌上清组)、CTL+Treg+50%SiHa上清培养组(共培养组)CTL+Treg+50%SiHa上清+8μg/mlβ-榄香烯干预组(β-榄香烯组)、CTL+Treg+50％SiHa上清+2μl/ml清毒栓醇提水沉液组(清毒栓组)五组中CTLA4、PD1的蛋白表达情况。临床研究选取北京中医药大学东方医院妇科门诊2009年8月～2011年1月就诊的宫颈HR-HPV感染的患者共90例。随机分成清毒栓治疗组、安达芬对照组、随诊观察组三组进行治疗观察3个月。用HCⅡ检测子宫颈HPVDNA含量(病毒负荷)的变化,判断清毒栓治疗高危型宫颈HPV感染的临床疗效,同时进行临床症状积分统计分析。结果1.随着宫颈上皮内瘤变病程的进展,转录因子T-be t阳性的Th1类细胞数量增多,各级病变中Thl类细胞数量变化的差异具有显著性(P<0.05)；转录因子GATA 3阳性的Th2类细胞数量减少,其中宫颈炎症与CINⅠ级病变中细胞数量变化差异不显著(P>0.05),各级CIN病变中Th2类细胞数量变化的差异具有显著性(P<0.05)；转录因子Foxp3阳性的Treg细胞数量增多,但在宫颈炎症、CINⅠ级及CINⅡ级病变中Treg细胞数量变化差异不显著(P>0.05),在CINⅢ级病变中,Treg细胞的数量较其他组显著增多(P<0.05)。2.应用CCK-8法测定单个核细胞(PBMC)的生长情况发现细胞在前48h处于生长停滞状态,根据细胞生长曲线,以下实验的检测宜在72h-120h内进行。3.不同浓度SiHa细胞培养上清液作用于CD8+CTL细胞,与空白血清对照组比较,75%培养上清组有统计学差异(P<0.05)；而作用于Treg细胞时,50%培养上清组和75%培养上清组与对照组比较,有统计学差异(P<0.05),故选择75%Siha上清液作为下面实验的干预浓度。4.免疫磁珠法分离人外周血CD8+T及CD4+CD25+T细胞,经流式细胞术检测CD8+纯度为87.37±0.93%,CD4+CD 25+T细胞纯度为89.12±2.17%。5.利用CFSE标记CTL,与Treg共培养4天后,CFSE呈单峰,未见明显增殖,而CTL单独培养4天后,可见CFSE呈双峰,即开始出现了细胞增殖,表明Treg对CTL增殖有明显抑制作用。6.4μg/ml.6μg/m 1.8μg/ml的β-榄香烯均可促进CTL增殖,选择较高的药物浓度8μg/ml作为后续实验的药物干预浓度。1μl/ml.1.5μl/m 1.2μ1/m l的清毒栓醇提水沉液均可促进CTL增殖,选择较高的药物浓度2μl/ml做为后续实验的药物干预浓度。7.经Western-Blot检测并测定电泳条带灰度值,CTL与Treg共培养组CTLA4、PD1的蛋白表达量明显增加,β-榄香烯及清毒栓干预后CTLA4、PD1的蛋白表达量显著降低,且清毒栓组蛋白表达量降低的更加明显。说明β-榄香烯、清毒栓都可以抑制Treg对CTL中CTLA4.PDl蛋白的上调作用,且清毒栓的作用更显著。8.清毒栓与安达芬都能有效的降低HC-ⅡHPVR2u/co比值即HPVDNA病毒负荷量,治疗前后自身对照结果有统计学意义(P<0.05),两组比较无显著性差异,与随诊观察组比较有统计学差异(P<0.05)；同时发现两种药物对临床症状、体征的改善作用明显,自身前后对照统计学差异极显著(P<0.01),与随诊观察组比较统计学差异也极显著(P<0.01)。结论1.宫颈病变局部免疫微环境中的Th1、Th2类细胞及Treg细胞都参与了宫颈上皮内瘤变的免疫反应,Treg细胞可能通过免疫抑制作用参与病变进展。2.清毒栓可以降低CTL中CTLA4、PD1蛋白的表达,可能是通过抑制Treg细胞的功能来降低CTLA4、PD1蛋白的表达,从而改变局部免疫微环境而达到抑制肿瘤的作用3.清毒栓能有效降低宫颈高危型人乳头瘤病毒(HR-HPV)感染的病毒负荷。并能有效改善临床症状、体征。更多还原

【Abstract】 Object:1. This research from the view of local lesion immune microenvironment, through researching the immunohistochemical expression of immune microenvironment related transcription factor T-bet, GATA3, Foxp3 in cervical intraepithelial neoplasia tissues, from the cellular level explored the relationship between cervical intraepithelial neoplasia progression and the number of Thl,Th2 and Treg in local lesion immune microenvironment.2. SiHa cervical cancer cells in the supernatant, CTL, Treg cell co-culture system as an object of study, observation of Treg, Qingdu tied to CTLA-4, PD-1 protein expression to explore the Treg cells in the tumor immune escape mechanism of traditional Chinese medicine suppository on disinfection effect local immune microenvironment.3. By detecting Qingdu Suppository, AnDafen and followed up for observation of the three groups observed after treatment viral load after 3 months and changes in clinical symptom score, judge drug suppository medicine clear clinical efficacy.Method:Empirical study:1. Select by HE staining, the pathological diagnosis of cervical inflammation and cervical intraepithelial neoplasia of the cervix 128 patients, divided into cervical inflammation, CINⅠgrade, CINⅡgrade and CINⅢgrade 4, in which cervical inflammation, CINⅡgrade lesions 32 Cases, CINⅠgrade of 34 cases and CINⅢgrade lesions of 30 cases.2. CCK-8 determination of the growth curve of PBMC.3. SiHa cells with different concentrations of supernatant role in CTL cells and Treg cells, flow cytometry CD8, Treg cell percentage changes.4. Isolation of human peripheral blood CD8+T cells, Treg cells, flow cytometry cell sorting the purity.5. CFSE labeling technique using cell value to understand the Treg cells to inhibit the function of CTL cells.6.4μg/ml,6μg/ml,8μg/ml ofβ-elemene could promote the proliferation of CTL, select a higher concentration of the drug as a follow-up experiments 8p.g/ml concentration of drug intervention. 1μl/ml,1.5μl/ml,2μl/ml the disinfection of water sink plug ethanol solution could promote the proliferation of CTL, select a higher drug concentration 2μl/ml follow-up experiments as the concentration of drug intervention7. Application of Western-Blot test and compare the CTL normal culture (control group), CTL 50% SiHa supernatants group (cancer supernatant group), CTL+Treg 50% SiHa supernatants group (training group), CTL+ Treg 50% SiHa supernatants+8μg/mlβ-elemene intervention group (β-elemene group), CTL+ Treg 50% SiHa supernatants+2μl/ml ethanol Qingdu suppository water precipitating solution group (Qingdu Suppository Group) five groups CTLA4, PD1 protein expression.Clinical research:1. The investigated group comprised 90 patients suffering from cervical HR-HPV infection, which all visited the DongFang Hospital of Beijing University of Chinese Medical, since Aug.2009 to Jan.2011. Randomly divided into treatment group Qingdu Suppository, AnDafen control group, followed up the observation group were treated three groups observed for 3 months. HCⅡdetected by cervical HPVDNA content (viral load) changes, to determine high-risk Qingdu suppository in the treatment of cervical HPV infection in clinical efficacy, but statistical analysis of the clinical symptom score.Results:1. With the progression of cervical intraepithelial neoplasia,the number of transcription factors T-bet positive Thl cells were significantly increased (P< 0.05).The number of transcription factors GATA3 positive Th2 cells were decreased, including Th2 celluar number change between cervical inflammation and CIN I was not significant P>0.05),the number changes among every grade of cervical intraepithelial neoplasia were significant (P<0.05).The number of transcription factors Foxp3 positive Treg cells were increased including Treg celluar number changes among cervical inflammation, CINⅠand CINⅡwere not significant (P>0.05),the number of Treg in CINIII was increased significantly (P<0.05)2. Application of CCK-8 determination of mononuclear cells (PBMC) found that the growth of cell growth and stagnation in the previous 48h, according to cell growth curve, the following experiment testing should be carried out in the 72h-120h.3. Supernatant of SiHa cells with different concentrations of the role of the CD8+CTL cells, compared with blank control group, serum,75% of the culture supernatant were significantly different (P<0.05); while the effect on Treg cells, 50% of the culture supernatant of group And 75% of the culture supernatant compared with the control group, there are significant differences (P<0.05),75% SiHa supernatant was chosen as the experimental intervention in the following concentrations.4. Isolation of human peripheral blood CD8+T and CD4+CD25+T cells by flow cytometry CD8+ purity was 87.37±0.93%, CD4+CD25+T cell purity was 89.12±2.17%.5. CFSE labeled by CTL,4 days after co-culture with Treg, CFSE showed a single peak, no significant proliferation, and CTL 4 days cultured alone, showing a bimodal CFSE, which began to appear in cell proliferation, suggesting that Treg proliferation of CTL inhibit.6.4μg/ml,6μg/ml,8μg/ml ofβ-elemene could promote the proliferation of CTL, select a higher concentration of the drug as a follow-up experiments 8μg/ml concentration of drug intervention.1μl/ml,1.5μl/ml,2μl/ml the disinfection of water sink plug ethanol solution could promote the proliferation of CTL, select a higher drug concentration 2μl/ml follow-up experiments as the concentration of drug intervention.7. Application of Western-Blot test and compare the CTL normal culture (control group), CTL 50% SiHa supernatants group (cancer supernatant group), CTL + Treg 50% SiHa supernatants group (training group), CTL + Treg 50% SiHa supernatants +8μg/mlβ-elemene intervention group (β-elemene group), CTL + Treg 50% SiHa supernatants+2μl/ml ethanol Qingdu suppository water precipitating solution group (clear Drug suppository group) five groups CTLA4, PD1 protein expression.8. Qingdu suppository and AnDafen can effectively reduce HC-ⅡHPVRlu co ratios that HPVDNA viral load, self-control results before and after treatment was statistically significant (P<0.05), showed no significant difference, and with the clinic observed group differ (P<0.05); also found that the two drugs on clinical symptoms and signs improved significantly effect (P<0.01), and followed up for observation group was significantly different (P<0.01).Conclusion:1. The local immune microenvironment in cervical lesions in the Th1, Th2 type cells and Treg cells are involved in cervical intraepithelial neoplasia of the immune response, Treg cells may progress through the immunosuppressive effects in disease2. Qingdu suppository can reduce the CTL in CTLA4, PD1 protein expression probably by inhibiting the function of Treg cells to reduce the CTLA4, PD1 protein expression, thus changing the local immune microenvironment to inhibit tumor effect.3. Qingdu suppository can reduce cervical high-risk human papillomavirus (HR-HPV) infection in viral load, also can effectively improve the clinical symptoms and signs.更多还原